Engineers and scientists in the United States, Canada, and China have developed a technology for investigating how the physical properties of cells within laboratory-grown 3D breast tumor models, or organoids, can drive the tumor to become invasive. Headed by researchers at the department of engineering, Massachusetts Institute of Technology (MIT), the team used a technology platform combining confocal microscopy with optical tweezers to demonstrate how cells in the interior of growing breast tumor organoids are small and stiff, while the cells on the periphery are softer and swollen because they contain more water. Their studies showed how these softer, peripheral cells are more able to stretch beyond the tumor body and form “invasive tips” that eventually are able to break away and spread to other sites, or metastasize.

The findings offer up new insights into tumor cell evolution and behavior that could point to new anticancer therapeutic strategies focused on changing the physical properties of cancer cells to delay or potentially even prevent a tumor from spreading. “You can think of the tumor like a sponge,” said research lead Ming Guo, PhD, assistant professor of mechanical engineering at MIT. “When they grow, they build up compressive stresses inside the tumor, and that will squeeze the water from the core out to the cells on the outside, which will slowly swell over time and become softer as well—therefore they are more able to invade.”

The scientists, including MIT first author Yu Long Han, and colleagues, described their results in Nature Physics, in a paper titled, “Cell swelling, softening and invasion in a three-dimensional breast cancer model.”

Living cells are dynamic systems that undergo a diverse range of processes, including gene expression and intracellular dynamics and forces at the molecular level, and cell contraction, deformation, and migration at the cellular level, the authors explained. “Within a multicellular system, the precise control of these physical characteristics in space and time is critical for the maintenance of mechanical integrity and biological function.” Different diseases, developmental disorders, and poor wound repair can result when such molecular- and cell-level mechanisms don’t all work together properly. Studying how these processes are maintained in a native, 3D multicellular microenvironment is a real challenge, however, and to date, “ … it remains unknown how the physical characteristics of individual cells regulate and coordinate tumor development and invasion,” the authors acknowledged.

Scientists have suspected that cancer cells migrating away from a primary tumor are able to do so partly because they are relatively soft and pliable, which allows them to navigate through the body’s vasculature to distant sites. While previous research has demonstrated the soft, migratory nature of individual cancer cells, Guo’s team developed an approach for studying the role of cell stiffness in the context of a developing tumor in 3D.

“People have looked at single cells for a long time, but organisms are multicellular, three-dimensional systems,” Guo said. “Each cell is a physical building block, and we’re interested in how each single cell is regulating its own physical properties, as the cells develop into a tissue like a tumor or an organ.”

The researchers used recently developed techniques to grow healthy human epithelial cells in 3D and transform them into a human breast cancer tumor in the lab. Over the course of a week, the researchers watched as the cells multiplied and coalesced into a benign primary tumor that comprised several hundred individual cells. They saw how, starting from a single cell, a multicellular cluster started to grow, and invasive branches developed over approximately 10 days.

The process starts with an individual cell proliferating to form a spherical cluster, which then grows into a larger spheroid with cells in the center, or core, and around the edge, or periphery. As the spheroid develops further, invasive branches extend from the main body into the surrounding extracellular matrix (ECM). “The phenotype observed in this 3D breast cancer model shows uncontrolled cellular proliferation, lack of cellular polarization, and the initiation of matrix invasion, much like those observed in vivo in invasive ductal carcinomas,” the investigators stated.

As part of their experimental approach, the researchers repeatedly infused the growing mass of cells with plastic particles. They then probed each individual cell’s stiffness using optical tweezers. The technique involves directing a highly focused laser beam at a cell. In this case, the team trained the laser on a plastic particle within each cell, pinning the particle in place. They then applied a slight pulse in an attempt to move the particle within the cell, akin to using tweezers to pick a piece of eggshell out from the surrounding yolk.

A tumor’s invasiveness depends on its water content and the stiffness of its exterior. In the top row, a tumor progresses normally toward an invasive profile. If water is drawn out of the same tumor (middle row), it is less invasive, compared to when the tumor is infused with water (bottom row), causing it to quickly burst and invade surrounding tissue. [Yu Long Han]
Guo says the degree to which a particle can be moved gives an indication of the stiffness of the surrounding cell. The more resistant the particle is to being moved, the stiffer a cell must be. The team’s experimental results indicated that the hundreds of cells within a single benign tumor exhibited a gradient of stiffness as well as size. The cells in the interior of the growing mass were smaller and stiffer, while the further the cells were from the core, the softer and larger they were. These peripheral cells also became more likely to stretch out from the spherical primary tumor and form branches, or invasive tips.

The researchers found that the cells at the tumor’s edges were softer because they contained more water than those in the center. The cells in the center of a tumor are surrounded by other cells that press inward, squeezing water out of the interior cells and into those cells at the periphery, through the nanometer-sized gap junction (GJ) channels between them.

To see whether altering the peripheral cells’ water content would affect their invasive behavior, the investigators added low-molecular-weight polymers to the tumor solution to draw water out from cells. They found that the outer cells then shrank, became more stiff, and were less likely to migrate away from the tumor—a measure that delayed metastasis. When water was added to dilute the tumor solution, the cells at the periphery swelled, became softer, and formed invasive tips more quickly.

The investigators then obtained a sample of a patient’s breast cancer tumor and measured the size of every cell within the tumor sample. They observed a gradient of size that was similar to that observed in the lab-derived tumor. Cells in the tumor’s core were smaller than those closer to the periphery. “Within the tumor mass, spheroidal, acinar clusters of cells surrounded by basement membrane were evident, which share similar characteristics with our 3D cancer model,” the scientists noted. “In such spheroidal acinar clusters, we found that the nuclear volume increased as the distance from the center increased, consistent with our model system. Cells in the core had smaller volumes, while invasive cells that appeared to have escaped from the main cluster had larger volumes.”

Early stage tumor
Early stage tumor. [MIT]
Late stage tumor
Late stage tumor. [MIT]
“We found this doesn’t just happen in a model system—it’s real,” Guo said. “This means we may be able to develop some treatment based on the physical picture, to target cell stiffness or size to see if that helps. If you make the cells stiffer, they are less likely to migrate, and that could potentially delay invasion.”

The role of gap junctions in cancer progression is a matter for debate, the authors pointed out, “with both promotion and suppression of invasiveness having been observed across various types of cancer and GJ.” They suggested that their results hint at a purely physical mechanism by which GJs can impact on cancer progression. “A change in the water content of cells and hence the degree of molecular crowding, will affect a wide range of downstream cell functions and properties … These mechanical changes are shown to arise from supracellular fluid flow through gap junctions, the suppression of which delays the transition to an invasive phenotype … As such, our emerging physical picture of tumor progression now includes 3D spatiotemporal evolution of cellular physical properties.”

Guo suggests that in the future clinicians may be able to look at a tumor and, based on the size and stiffness of cells from the inside out, be able to say with some confidence whether a tumor will metastasize or not. “If there is an established size or stiffness gradient, you can know this will cause trouble,” Guo says. “If there’s no gradient, you can maybe safely say it’s fine.”

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